The present invention relates generally to agents useful for stimulating the immune system of a host animal, and more particularly, to agents and methods for providing selective stimulation to the immune system of a host animal.
A primary function of the immune system is to protect the animal against the deleterious effects of invading pathogens. One type of immune system response to such invasion, known as cell-mediated immune response, protects the animal against invasion by microorganisms, such as bacteria, protozoa and viruses, and also against invasion by abnormal and malignant cells. Cell-mediated immune response is controlled by the T-lymphocytes, or T-cells. When the body recognizes the presence of an invading pathogen, two types of T-cells are produced, namely cytotoxic, or killer, T-cells that can destroy the invading pathogen, and helper T-cells that enhance the body""s defenses against the invader.
The immune system of an animal may become compromised due to such factors as disease, exposure to etiologic agents, allergic reactions, autoimmune system disorders and advanced age. When the animal becomes immunocompromised in this manner, the ability of the T-cells to destroy invading pathogens is reduced, and in severe cases, may be lost altogether. In addition, even when an animal has a functional immune system, the ability of the T-cells to resist infection by certain pathogens may be insufficient.
When the invading pathogen is a microorganism, such as a bacterium, it is common to administer antibiotics to the animal suffering from such invasion. The purpose of the antibiotics is to control the spread of the bacteria within the body, and preferably, to destroy it altogether. Many effective antibiotics have been developed to treat both humans and non-human animals against diseases caused by invading etiologic agents. The effectiveness of these antibiotics has contributed to their widespread usage. However, this widespread usage of antibiotics has caused increased concern in recent years that antibiotic-resistant strains of bacteria may develop and spread. As a consequence, regulatory authorities have begun to more closely scrutinize and monitor the use of certain antimicrobial agents. In some cases, certain uses of these agents have been banned. In order to minimize the possibility that resistant bacterial strains may develop, it would be desirable to reduce the use of these antimicrobial agents. However, any such reduction would necessitate the development of new treatments to counter the harmful effects on the body that may be caused by an etiologic agent if left untreated.
It is believed that the class of drugs known as immunopotentiators acts on the immune system by priming the function of leukocytes, thereby enabling the leukocytes to respond with increased bactericidal activity upon stimulation by a pathogen. The inventors postulate that the mechanism of action of the immunopotentiators in the present application is a blastogenic effect on the T-cells, which is mediated by cytokines. The cytokines then prime the function of bactericidal leukocytes, such as macrophages, killer T-cells and polymorphonuclear leukocytes (PMNs). In the presence of pathogenic bacteria, the leukocytes phagocytize and kill the bacteria.
It is well known that certain bacterial agents are the source of numerous diseases afflicting animals. An example of an agent affecting both human and non-human animals is the bacterium Escherichia coli. Outbreaks caused by various strains of E. coli have been widely documented. Such outbreaks in humans are known to result from, among others, the consumption of undercooked and/or unwashed food. These outbreaks have been known to cause severe intestinal distress and, in some instances, death. The exposure to E. coli is also known to cause severe problems in many animal species. For example, the syndrome avian colibacillosis is caused in poultry by E. coli. Manifestations of colibacillosis in poultry may include acute septicemia, airsacculitis, pericarditis, perihepatitis and peritonitis. The control of this disease is economically important to poultry producers, because it causes morbidity, mortality, lack of uniformity, decreased performance and increased condemnations.
Etiologic agents such as E. coli are often secondary pathogens to a primary viral insult. However, some virulent bacterial isolates are known to cause disease even in the absence of a primary insult. E. coli causes airsacculitis in poultry by colonizing in the air sac following a reduction in respiratory host defenses, which is often a sequelae to stressors or viral infections, such as infectious bronchitis virus and Newcastle""s disease virus. Presently, colibacillosis in poultry is prevented by the early administration of antimicrobial agents such as cephalosporin, quinolone, and aminoglycoside antibiotic to one-day old chicks. However, in view of the concern that use of these antimicrobial agents may contribute to the development of antibiotic-resistant bacteria, it is desirable to reduce such use.
In addition, due to the crowded pens and unsanitary structures that are often used to house non-human animals, these animals are at a high risk of infection and re-infection, and of immune system disorders occurring as a result of such conditions. In many cases, an infected and/or immunocompromised animal must be treated with antibiotics in order to prevent and/or attempt to control the disease.
Accordingly, it is desired to provide a class of compounds that stimulates the natural immune system of a host animal, thereby enabling the host animal to increase its resistance to infection. In addition, it is desired to provide a class of compounds that provides an alternative to the use of antibiotics.
A class of drugs, known as immunopotentiators, is provided. These drugs, when introduced into the body of a host animal, selectively stimulate the natural immune system of the host, thereby reducing or eliminating altogether the necessity to introduce antibiotics into the host""s body in order to fight certain infectious agents. As a result of this selective stimulation, i.e., T-cell blastogenesis and production of cytokines, the natural immune system of the host animal achieves an enhanced ability to control the bacteria.
In one aspect of the present invention, there is provided an immunopotentiating composition comprising a physiologically acceptable carrier and an effective immunopotentiating amount of a compound defined by the following Formula I: 
wherein:
R1 is H, C1-C4 alkyl or (xe2x95x90O);
R2 is C(O)R8, CH2C(O)R8, CN, CH2OH, OH, OC(O)R9 or OS(O)2R9 
R3, R4 and R5 are each independently H or C1-C4 alkyl;
R6 is C(O)OCH2Ar3, C(O)NHCH2Ar3, C(O)Ar1, C(O)CH(R3)Ar2, C(O)CH(R3)CH2Ar2, C(O)CH(R3)CH2CH2Ar4 or C(O)NHR9;
R7 is H or CH2C(O)R8;
R8 is H, C1-C4 alkyl, OR10, NHR11 or OH (when R6 is C(O)Ar, or C(O)CH(R3)Ar2);
R9 is C1-C4 alkyl;
R10 is C2-C8 alkyl, CH2CH2N(CH3)2 or CH3 (when R6 is C(O)Ar1 and Ar1 is phenyl);
R11 is C2-C6 alkyl, CH2CH2N(CH3)2 or C7-C9 alkyl (when R6 is C(O)Ar1);
Ar1 is phenyl, 4-fluorophenyl, 2-thienyl, 3-thienyl or 1,4-biphenyl;
Ar2 is phenyl or 2-thienyl;
Ar3 is phenyl;
Ar4 is 2-thienyl;
Ar5 is phenyl or 2-halo-phenyl;
halo is Cl, Br or F; and
Ar6 is plienyl or 4-fluorophenyl;
with the proviso that when R7 is CH2C(O)R8, then R2 and R3 are H, R4 and R5 are H or C1-C4 alkyl; R6 is C(O)CH(R3)Ar4; R8 is H, C1-C4 alkyl, OR9 or NHR10; R9 is C1-C8 alkyl and R10 is C2-C8 alkyl; and with the further proviso that when R1 is (xe2x95x90O), then R2 is C(O)R8 or CH2C(O)R8; R3, R4 and R5 are H or C1-C4 alkyl; R6 is C(O)OCH2Ar3, C(O)Ar6 or C(O)NHR9; R7 is H or, together with R5, CH2CH2: R8 is H, C1-C4 alkyl, OR10 or NHR11; R9 is C1-C4 alkyl; R10 is C1-C8 alkyl and R11 is C2-C8 alkyl; or Formula II: 
wherein:
R12 is C(O)R18 or CH2C(O)R18;
R13, R14, R15 and R16 are each independently H or C1-C4 alkyl;
R17 is C1-C4 alkyl;
R18 is H, C1-C4 alkyl; OR19; NHR20 or N(CH3)2;
R19 is C1-C8 alkyl;
R20 is C2-C8 alkyl;
Ar7 is phenyl, 4-fluorophenyl or 1,3-thiazol-2-yl;
X is I, Cl, Br, O(SO2)R21; and
R21 is CH3, CF3, phenyl or p-methylphenyl;
or a physiologically acceptable salt of the compound of Formula I or Formula II.
In addition to the foregoing, another aspect of the invention is a method for potentiating the immune system of a host animal comprising administering to the host the immunopotentiating composition defined above. Yet another aspect of the invention is a method for protecting a host animal against infection, comprising administering to the host the immunopotentiating composition defined above.
A still further aspect of the invention is directed to certain novel compounds, and processes for preparing the novel compounds. The novel compounds are described by Formulas I and II as provided above, with the following additional provisos: when R1, R4, R5 and R7 are H, R2 is C(O)R8, R6 is C(O)Ar3 and R8 is OR10, then R3 is not C1 alkyl and R10 is not C1-C2 alkyl; when R1 is (xe2x95x90O), R2 is C(O)R8, R4, R5 and R7 are H, R6 is C(O)Ar6, R8 is OR10 and Ar6 is phenyl, then R3 is not C2 alkyl or C4 alkyl and R10 is not C1-C2 alkyl; when R1 is (xe2x95x90O), R2 is C(O)R8, R3, R4, R5 and R7 are H, R6 is C(O)OCH2Ar6, R8 is OR10 and Ar6 is phenyl, then R10 is not C1-C2 alkyl; when R1, R3, R4, R5 and R7 are H, R2 is C(O)R8, R6 is C(O)CH2Ar3 and R8 is OR10, then R10 is not C2 alkyl; and when R1, R2, R3, R4 and R5 are H, R6 is C(O)Ar5, R7 is CH2C(O)R8 and Ar5 is phenyl, then R8 is not C1 alkyl or NHR10 (when R10 is C1 alkyl).
The present invention is directed to the use of certain compounds, known as immunopotentiators, to stimulate, or potentiate, the immune system of a host animal. More particularly, the invention relates to immunopotentiating compositions, and to a method for potentiating the immune system of a host animal having an immune system in need of potentiating, by administering to the host animal an effective amount of one or more immunopotentiating compounds represented generally by Formula I or Formula II, as defined and limited above, or a physiologically acceptable salt thereof.
The invention is also directed to a method for protecting a host animal against infection, and to certain novel compounds, as further defined and limited above.
As used herein, the chemical terms have their usual meanings unless otherwise indicated. For example, the term xe2x80x9calkylxe2x80x9d by itself or as part of another substituent, unless otherwise indicated, includes straight or branched aliphatic hydrocarbon radicals, such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, pentyl, hexyl, heptyl, octyl and nonyl groups having the indicated number of carbon atoms.
The term xe2x80x9cArxe2x80x9d refers to an aromatic or heteroaromatic group, or a substituted aromatic or heteroaromatic group.
The terms xe2x80x9cimmunopotentiatorxe2x80x9d, xe2x80x9cimmunopotentiating agentxe2x80x9d and xe2x80x9cimmunopotentiating compoundxe2x80x9d refer to an agent or compound capable of stimulating, enhancing or potentiating normal immune function, or restoring, stimulating, enhancing or potentiating a depressed immune function, or both.
The term xe2x80x9canimalxe2x80x9d includes all living organisms, exclusive of plants, insects and bacteria, having a responsive immune system and having T-cells capable of undergoing blastogenesis. The term is most particularly intended to include, but not be limited to, farm animals, such as all avian, bovine, ovine and porcine species; other mammals such as humans, non-human primates, canines and felines; and reptiles and fish.
The terms xe2x80x9ceffective amountxe2x80x9d and xe2x80x9ceffective immunopotentiating amountxe2x80x9d of a compound refer to the amount of that compound that will restore immune function to near normal levels, or increase immune function above normal levels in order to control or reduce infection.
A xe2x80x9cphysiologically acceptable saltxe2x80x9d is a salt of a compound of the above-referenced Formula I or Formula II that is substantially non-toxic to a host animal.
The immunopotentiator compounds of the present invention have been found to enhance the blastogenesis of peripheral blood T-lymphocytes derived from avian, bovine, porcine, canine and non-human primates in vitro. Some of the immunopotentiator compounds used in the present invention have been found to be active in vitro at concentrations as low as 1 ng/ml. In many cases, the compounds are non-cytotoxic in vitro at concentrations up to 100 xcexcg/ml, and in some cases, up to 1 mg/ml. Many compounds have been found to provide a rapid blastogenic response in vitro within 72 hours, and some have provided this response within 48 hours.
The present invention is further directed to certain novel compounds defined above, and subject to the specified limitations. The novel compounds, in most instances, are useful as immunopotentiating agents. Some of the novel compounds are useful as synthetic intermediates in the preparation of other immunopotentiating agents. In some cases, the immunopotentiator agents described for use in the inventive method are known compounds.
Although the invention is not so restricted, many of the compounds of Formula I of the present invention may be prepared by one of the synthetic routes described below, each of which involves the acylation of a starting amine. The starting amines utilized in these processes are either commercially available, or may be readily prepared by hydrogenation and, if necessary, subsequent oxidation of a corresponding pyridine. Generally speaking, the compounds of Formula I may be prepared by acylating an amine compound of Formula III 
with a compound of Formula IV
R6Yxe2x80x83xe2x80x83IV
wherein R1, R2, R3, R4, R5, R6 and R7 are as previously defined; and Y is Cl, Br, or OH.
One synthetic route involves the dissolution of the appropriately substituted starting amine in dichloromethane. 1.1 molar equivalents of commercially available diisopropylethylamine (2.1 molar equivalents when the starting amine is in the salt form) is added to the mixture. Excessive temperature rise is controlled with an ambient temperature water bath (or ice water bath on larger scale reaction). 1.0 molar equivalents of the appropriately substituted acid chloride is then added slowly to control any rise in reaction temperature. After the addition of the acid chloride is completed, the mixture is allowed to stir at ambient temperature for approximately 1-4 hours. The mixture is then diluted with fresh dichloromethane, and washed with either 1 N HCl or water. The dichloromethane solution is then dried with anhydrous potassium carbonate and evaporated at ambient temperature under reduced pressure. The crude material is purified by high-vacuum short-path distillation, chromatography over silica gel, or reversed phase HPLC.
Another synthetic route involves suspending the appropriately substituted carboxylic acid in dichloromethane, and thereafter adding 1.0 molar equivalent of commercially available 1-(3-dimethylaniinopropyl)-3-ethylcarbodiimide hydrochloride, or alternatively, 1,3-dicyclohexylcarbodiimide (DCC). The mixture is allowed to stir at ambient temperature for approximately 1 hour. 1.0 molar equivalent of the appropriately substituted amine or alcohol is then added, and the mixture is stirred at ambient temperature for approximately 18 hours. The reaction mixture is diluted with fresh dichloromethane and then washed with water. The dichloromethane is dried with anhydrous potassium carbonate and evaporated at ambient temperature under reduced pressure. The crude material is then purified by either high-vacuum short-path distillation, chromatography over silica gel, or reversed phase HPLC.
Yet another synthetic route may be used when a urea is to be produced. In this case, the appropriately substituted starting amine is dissolved in dichloromethane, and 1.0 molar equivalent of the desired isocyanate is added. The mixture is then stirred at ambient temperature for approximately 3 hours. The mixture is diluted with fresh dichloromethane, and washed with water. The dichloromethane is dried with anhydrous potassium carbonate and evaporated at ambient temperature under reduced pressure. The crude material is then purified by either high-vacuum short-path distillation, chromatography over silica gel, or reversed phase HPLC.
The products obtained by the respective synthetic routes may then be further manipulated by means such as hydrolysis, alkylation, further acylation, reduction or oxidation to provide the desired analog. If desired, the compounds can be resolved by either utilizing chirally-resolved starting materials or by chiral resolution of the finished product.
Many of the compounds of Formula II may be prepared by either of the synthetic routes described below. Once again, the starting amines are either commercially available or may be prepared by the hydrogenation of a corresponding pyridine. Generally speaking, the compounds of Formula II may be prepared by alkylating an amine compound of Formula VII 
with a compound of Formula V
Ar7CH2Xxe2x80x83xe2x80x83V
or, reductively aminating the amine compound of Formula VII with a compound of Formula VI
xe2x80x83Ar7CHOxe2x80x83xe2x80x83VI
and, quaternizing the resulting amine compound with a compound of Formula VIII
R17Xxe2x80x83xe2x80x83VIII
wherein R12, R13, R14, R15 R16, R17, Ar7 and X are as previously defined.
One synthetic route involves the alkylation and quaternization of the amine. In this process, the appropriately substituted starting amine is dissolved in dichloromethane. 1.1 molar equivalents of diisopropylethylamine is added followed by addition of an appropriately substituted bromide reagent. After the addition is complete the mixture is allowed to stir at ambient temperature for approximately 18-24 hours. The mixture is then evaporated at ambient temperature under reduced pressure and the crude product is purified by either high-vacuum short-path distillation, chromatography over silica gel, or reversed phase HPLC. This material is then dissolved in anhydrous diethyl ether and a large excess of methyl iodide is added. The mixture is then refluxed for 20-24 hours and resulting desired product precipitate is isolated by filtration.
Another synthetic route involves the reductive amination and quaternization of the amine. In this process, the appropriately substituted starting amine is dissolved in anhydrous methanol. 1.0 molar equivalents of commercially available aldehyde is added, followed by 1.9 molar equivalents of commercially available sodium cyanoborohydride. The mixture is then allowed to stir at ambient temperature for 20-24 hours. The mixture is diluted with dichloromethane and washed with water. The organic phase is separated, washed with saturated sodium chloride solution, dried with anhydrous potassium carbonate, and evaporated at ambient temperature under reduced pressure. The crude product is purified by either high-vacuum short-path distillation, chromatography over silica gel, or reversed phase HPLC. This material is then dissolved in anhydrous diethyl ether and a large excess of methyl iodide is added. This mixture is refluxed for 20-24 hours, and resulting desired product precipitate is isolated by filtration.
The products obtained by the synthetic routes for preparing the compounds of Formula II may be further manipulated by means such as hydrolysis, alkylation, further acylation, reduction or oxidation to provide the desired analog. Also, if desired, the compounds can be resolved by either utilizing chirally-resolved starting materials or by chiral resolution of the finished product.
Specific examples of the use of the synthetic schemes described above to prepare compounds of Formulas I and II are provided herein in preparative Examples 1-73. Preparative Examples 1-73 further illustrate processes for the preparation of many of the compounds of the present invention. It is not intended that the invention be limited in scope by reason of any of the examples. Certain compounds listed below can be used to prepare other compounds, as further described in the preparative examples.
Unless otherwise stated in the preparative examples, the starting materials and chemicals used to prepare the compounds of the present invention and the compounds employed in the present invention are either commercially available or are readily prepared by known processes.
The terms and abbreviations used in the examples have their normal meanings unless otherwise designated. For example, xe2x80x9cxc2x0 C.xe2x80x9d refers to degrees Celsius; xe2x80x9cNxe2x80x9d refers to normal or normality; xe2x80x9cmmolxe2x80x9d refers to millimole; xe2x80x9ckgxe2x80x9d refers to kilograms; xe2x80x9cgxe2x80x9d refers to grams; xe2x80x9cmgxe2x80x9d refers to milligrams; xe2x80x9cxcexcgxe2x80x9d refers to micrograms; xe2x80x9cngxe2x80x9d refers to nanograms; xe2x80x9cmlxe2x80x9d refers to milliliter; xe2x80x9cMxe2x80x9d refers to molar; and xe2x80x9cMSxe2x80x9d refers to mass spectrometry.